Development of Microsatellite Loci for the Western Slimy Salamander (Plethodon Albagula) Using 454 Sequencing

Conservation Genet Resour DOI 10.1007/s12686-012-9784-5

TECHNICAL NOTE

Development of microsatellite loci for the western slimy salamander (Plethodon albagula) using 454 sequencing

Brett N. Spatola • William E. Peterman • Nathan T. Stephens • Grant M. Connette • Donald B. Shepard • Kenneth H. Kozak • Raymond D. Semlitsch • Lori S. Eggert

Received: 7 September 2012 / Accepted: 28 September 2012 Springer Science+Business Media Dordrecht 2012

Abstract Terrestrial salamanders of the genus Plethodon Plethodon are closely tied to cool, moist abiotic conditions, are closely tied to abiotic environmental habitat features making them particularly sensitive to habitat alteration and and are particularly sensitive to land use and habitat fragmentation (Marsh et al. 2008; Petranka 1998). Western change. To better understand the effects of land use and slimy salamanders (Plethodon albagula) inhabit forested habitat fragmentation on population genetic characteristics, areas throughout the Ozark Highlands (Conant and Collins we screened 137 primers developed from 454 sequence 1998). The ability to disperse through and persist in frag- libraries, and developed 27 microsatellite primers for the mented landscapes can have a great impact on genetic western slimy salamander, Plethodon albagula. Twenty- diversity among populations (Noe¨l et al. 2007), and an two of these primers cross-amplified in P. shermani. These understanding of gene flow across the landscape is critical new loci will be invaluable tools to study habitat variables to forming sound conservation and management decisions affecting gene flow, population genetic structure, and (Storfer et al. 2010). genetic diversity across heterogeneous landscapes. Samples from P. albagula were collected by taking 0.5 cm of tail tissue from salamanders at Danville (n = 51) and Keywords Plethodon shermani Population genetics Reform (n = 50) Conservation areas, Missouri, USA, which Landscape genetics are separated by *25 km. Samples were stored in 95 % EtOH at -20 C prior to DNA extraction. DNA was extracted using the Wizard SV 96 Genomic DNA Purification Habitat loss and fragmentation are key factors affecting the System (Promega, Madison, WI, USA). Sequences were stability of amphibian populations and can negatively generated using 454 technology (Roche, Branford, CT, USA) affect dispersal and population connectivity (Cushman in two ways; a microsatellite-enriched library was developed 2006; Stuart et al. 2004). Salamanders of the genus using methods outlined by Lance et al. (2010)forP. albagula, while traditional whole genome sequencing (WGS) was used to generate sequences for P. glutinosus, a sister species to B. N. Spatola W. E. Peterman (&) G. M. Connette P. albagula.TheP. albagula tissue was collected in Garland R. D. Semlitsch L. S. Eggert County, Arkansas , USA, approximately 500 km from the Division of Biological Sciences, University of Missouri-Columbia, 110 Tucker Hall, Columbia, sources of the samples were screened. For the WGS sequence MO 65211, USA data for P. glutinosus,weusedRepeatMaskerwithTandem e-mail: [email protected] Repeats Finder (Smit et al. 2012; Benson 1999) to identify reads containing microsatellites and then used a custom N. T. Stephens Department of Biological Sciences, Arkansas State University, PerlScript to identify reads with3 40 bp of sequence flanking Jonesboro, AR 72401, USA each side of the microsatellite region. We used Primer3 implemented within MSATCOMMANDER to identify and D. B. Shepard K. H. Kozak design primers for screening (Faircloth 2008; Rozen and Bell Museum of Natural History and Department of Fisheries, Wildlife, and Conservation Biology, University of Minnesota, Skaletsky 2000). Primers for 137 di-, tetra-, and penta- St. Paul, MN 55108, USA nucleotide microsatellites were developed from P. albagula 123 Conservation Genet Resour

Table 1 Primer sequences, repeat motif, multiplex pool, and concentration in reaction (lM) for 27 microsatellite loci for P. albagula Locus Primer sequence 50 to 30 Motif Label Multiplex Concentration GenBank #

PG_184b F: GTGCCCAGTGTGCTTTACAA AACAT(6) NED 1 0.1 JX946151 R: TTTGGGTCAAACCCTCTTTG PG_3XI F: AGCGGTGGATAGTCGTACAC AACAT(12) NED 2 0.05 JX946152 R: ATAGCACATAGGCAGATCAGTC PG_43M F: AGTCATTGTCAGCTTGCGC AATG(13) FAM 2 0.05 JX946153 R: GGGAGCTTGCATCAGGAAAG PG_P0G F: ACCTGTATTTCACGCTGCAC AATG(11) VIC 1 0.05 JX946154 R: CTGCACCTCTCACCCTACTG PG_OLQ F: AACTGCGGATAGTGGTCACC AACAT(10) – – – JX946155 R: AAGCAAACCAATGTCCCTGC PG_QWZ F: TCGTCTGATTATTGCGCTGC AATG(8) PET 2 0.05 JX946156 R: ACCTATCTCATCCACCACTGC PG_RIH F: AGCAGAGGGTTAGGGTATCG AAAG(16) FAM 2 0.05 JX946157 R: GCGCTCCGACACCTAAGG PG_V58 F: CTGTGCCACCTTGTTTCCTG AATG(10) PET 1 0.075 JX946158 R: TTGTGAGTCTCCTGCCCTTG PLAL_084 F: ACTCCACAAACTCACTACCTG ACAG(11) VIC 1 0.1 JX946132 R: TGTGGACCCTATTCTTGGCC PLAL_124 F: TCTCTCTAGCCTGACGTTCTG AG(12) NED 2 0.1 JX946133 R: TGCCGGGAATAAGACAAAGC PLAL_127 F: ATGTCCGAGCTATGAAACCC AATG(6) VIC 2 0.05 JX946134 R: GCACTCGCCTTGACCATTAC PLAL_190b F: AGAAGCTGTTGGCATGAGGT AGAT(26) FAM 1 0.1 JX946135 R: AGGTGCTATGGTGTGGCTTC PLAL_241 F: CAAAGGTAGGCAATGGTCTCG AATG(15) NED 2 0.1 JX946136 R: ACGAGCTAGACCTCTATTTGGG PLAL_315 F: CGGTGAATAACTCTTAAACCGC AGAT(20) NED 1 0.1 JX946137 R: TTAGCGTCATGTTGCCTCTG PLAL_331b F: GCTATAATGGCGGACAATGG AGAT(14) FAM 1 0.1 JX946138 R: TTTTCTAGTCGCCAGGATGC PLAL_402 F: AGTGGTGAGGGAGATGGATG ATCC(14) FAM 1 0.1 JX946139 R: TGGACTGTTGCTTTCTTGTGC PLAL_542 F: ATGCCTTAGGACCGCAGTAG AGAT(32) PET 1 0.1 JX946140 R: TGGGTTTCCTGGCATACTCC PLAL_545b F: TGGGCCTGGAGCATTACATA AGAT(27) VIC 2 0.1 JX946141 R: GCTTAGTGCAAGGTGTCTTCC PLAL_615 F: CCTAAGAGCACGGGACAGAG AGAT(14) VIC 2 0.1 JX946142 R: TATGAGGTCGATCGGTGAGC PLAL_701 F: CATGCGTACAGGATTAGGTCAG ACAG(16)…AGAT(16) FAM 2 0.1 JX946143 R: CAGTCTGCCTCTTTGTAAGGC PLAL_791 F: GCTTGTTTACTTGATGGAGC AATG(11) VIC 1 0.075 JX946144 R: ACGTAATCCCAGGTTACACTCC PLAL_815b F: GCTTCGTGCATTAAGTCGTG AATG(8) – – – JX946150 R: GATGCCACCGTTATTACCA PLAL_909 F: ATCGGAAGTGGGACCATAATG AACT(21) VIC 1 0.05 JX946145 R: TCGGAAGTGGGACCATAATG PLAL_AOYX3 F: AAATGGTTTCAGGCTGGCTC AATG(11) – – – JX946149 R: GGGACTGCAAAGTGACACATG

123 Conservation Genet Resour

Table 1 continued Locus Primer sequence 50 to 30 Motif Label Multiplex Concentration GenBank #

PLAL_B8DRY F: TGTGGACAGTGGGATCAAGG AGAT(10) VIC 2 0.1 JX946146 R: TGATGGAGCCAGAGAGCATG PLAL_C8W7K F: AGGACCTTGGCACTCTTAGG AGAT(9) PET 2 0.05 JX946147 R: CAGAGCGTCATTTCACCCTG PLAL_EIXNY F: TTTGTTTGTGGCTAGTCGTG ACAG(9)…AGAT(11) PET 2 0.15 JX946148 R: GCCCAGTCCTTGCTTCTTTC

Table 2 Size ranges, number of samples successfully ampliﬁed (N), number of alleles (NA), observed (HO) and expected (HE) heterozygosities, for microsatellite loci in P. albagula and P. shermani Locus P. albagula P. shermani

Size (bp) N NA HO HE Size (bp) N NA HO HE

PG_184b 182–192 99 2 0.475 0.436 166–206 8 6 0.750 0.817 PG_3XI 130–156 101 2 0.039 0.038 120–130 8 3 0.500 0.575 PG_43M 105–125 101 4 0.378 0.347 100–138 8 7 0.750 0.742 PG_OLQ ?????164–204 8 2 0.375 0.508 PG_POG 229–245 101 5 0.308 0.378 220–264 7 8 0.625 0.900 PG_QWZ 152–156 101 2 0.256 0.294 152–160 8 2 0.250 0.233 PG_RIH 204 101 1 0.000 0.000 190–204 8 2 0.125 0.125 PG_V58 140–156 101 3 0.307 0.399 132–168 8 6 0.375 0.792 Plal_084 345–413 101 3 0.29 0.324 245–421 8 10 0.625 0.950 Plal_124 302–358 101 8 0.593 0.704 240–294 8 10 1 0.95 Plal_127 87–103 101 3 0.436 0.512 84–116 8 4 0.625 0.692 Plal_190b 272–380 95 18 0.673 0.832 307–413 8 9 0.875 0.917 Plal_241 211–247 101 7 0.455 0.424 190–230 8 8 0.875 0.908 Plal_315 281–399 98 19 0.745 0.827 243–297 8 10 0.875 0.933 Plal_331b 166–186 101 6 0.653 0.638 129–177 8 7 0.875 0.867 Plal_402 107–147 101 7 0.394 0.417 59–147 8 7 0.750 0.850 Plal_542 200–248 101 7 0.652 0.65 138–231 8 10 0.750 0.917 Plal_545b 326 101 1 0.000 0.000 – – – – – Plal_615 165–181 101 4 0.2 0.208 – – – – – Plal_701 302–356 100 11 0.8 0.81 – – – – – Plal_815b ?????213–253 8 7 0.875 0.850 Plal_791 106–122 101 4 0.229 0.232 – – – – – Plal_909 156–192 101 7 0.632 0.676 122–182 8 7 1.000 0.867 Plal_AOYX3 ?????405–453 8 9 0.625 0.917 Plal_B8DRY 245–281 100 7 0.344 0.444 238–358 8 9 0.500 0.908 Plal_C8WYK 193–237 101 7 0.339 0.383 164–292 8 12 0.875 0.958 Plal_EIXNY 265–381 95 21 0.643 0.92 – – – – – Meana 100.14 7.14 0.447 0.495 7.95 7.05 0.676 0.781 a Excluding monomorphic loci. ‘‘?’’ signifies loci that worked in P. albagula, but could not be fit into multiplex reactions; ‘‘-’’signifies loci with no or poor amplification in P. shermani

(n = 87) and P. glutinosis (n = 50) sequences and screened dNTP, 0.5 units AmpliTaq Gold DNA polymerase (Applied using a single sample of total genomic DNA from Biosystems, Foster City, CA, USA), 0.89 bovine serum P. albagula. PCR conditions were standardized to 15 lL albumin (BSA), 0.4 lM of forward and reverse unlabeled containing 19 PCR Gold buffer, 2.0 mM MgCl2,2.0mM primer, and 1.0 lL (50 ng) genomic DNA. The PCR proﬁle

123 Conservation Genet Resour included an initial denaturing step at 95 C for 10 min, fol- understanding of ecology, population biology, and con- lowed by 35 cycles of 95 C denaturing for 45 s, 60 C servation of these species. annealing for 45 s, extension at 72 C for 45 s; and a final 5-min extension at 72 C. PCR products were separated in a Acknowledgments We thank C. Cramer (ASU) for sharing 454 4 % agarose gel with Gel Star (Lonza) and 100 bp ladder. sequences for P. albagula, and funding from the University of Missouri Research Board (CB000402), the DoD Strategic Environmental Forty primers yielding clear PCR products were further Research and Development Program (RC2155), the Arkansas Biosci- screened for polymorphism on seven samples. A total of 27 ences Institute (research component of Arkansas Tobacco Settlement polymorphic loci were identified and forward primers were Proceeds Act of 2000), National Science Foundation (DEB-0949590 to fluorescently labeled (Table 1). Primers were divided into KHK), and S.E. Trauth for Arkansas field site access. two multiplexes (Table 1) using Multiplex Manager (Holle- ley and Geerts 2009) and screened in 101 P. albagula samples References from the two conservation areas. Multiplex PCR reactions were done with the Qiagen Multiplex PCR kit (Valencia, CA, Benson G (1999) Tandem repeats finder: a program to analyze DNA USA) in an 8 lL volume using the following cycling condi- sequences. Nucleic Acids Res 27:573–580 tions: initial denaturing at 95 C for 15 min, followed by 35 Collins JT, Taggart T (2009) Standard common and current scientific cycles of 94 C denaturing for 30 s, annealing at 60 Cfor names for North America amphibians, turtles, reptiles, and crocodilians, 6th edn. http://www.cnah.org/pdf_files/1246.pdf. 135 s, and 72 C extension for 60 s; and a 30-min extension Accessed 17 Aug 2012 at 60 C. Amplification products were sized on an ABI 3730 Conant R, Collins JT (1998) Peterson field guides: reptiles and DNA Analyzer (Applied Biosystems) using Liz 600 size amphibians of Eastern/Central North America. Houghton Mifflin Company standard at the University of Missouri DNA Core Facility, Cushman SA (2006) Effects of habitat loss and fragmentation on and results were scored using GENEMARKER version 1.97 amphibians: a review and prospectus. Biol Conserv 128:231–240 (Softgenetics, State College, PA, USA). We calculated Faircloth BC (2008) MSATCOMMANDER: detection of microsatel- expected and observed heterozygosities using the Excel lite repeat arrays and automated, locus-specific primer design. Mol Microsatellite Toolkit (Park 2001), Hardy–Weinberg equi- Ecol Resour 8:92–94. doi:10.1111/j.1471-8286.2007.01884.x Holleley CE, Geerts PG (2009) Multiplex Manager 1.0: a cross- librium and linkage disequilibrium using GENEPOP version platform computer program that plans and optimizes multiplex 4.0.10 (Rousset 2008), and pairwise differentiation between PCR. Biotechniques 46:511–517 population pairs in GENODIVE version 2.0b22 (Merriman Lance S, Light J, Jones K, Hagen C, Hafner J (2010) Isolation and and VanTienderen 2004). Lastly, we cross-amplified all of characterization of 17 polymorphic microsatellite loci in the kangaroo mouse, genus Microdipodops; (Rodentia: Heteromyi- our primers in eight specimens of the IUCN vulnerable spe- dae). Conserv Genet Resour 2:139–141 cies P. shermani using PCR conditions described for initial Marsh DM, Page RB, Hanlon TJ, Corritone R, Little EC, Seifert DE, primer screening. Cabe PR (2008) Effects of roads on patterns of genetic Plethodon cinereus Alleles per locus for P. albagula ranged from 1 to 21 differentiation in red-backed salamanders, . Conserv Genet 9:603–613 (mean = 7.14, Table 2) and the observed heterozygosity Merriman PG, VanTienderen PH (2004) GENOTYPE and GENO- ranged from 0.039 to 0.800 (mean = 0.447). The popula- DIVE: two programs for the analysis of genetic diversity of asexual organisms. Mol Ecol Notes 4:792–794 tions were significantly differentiated (FST = 0.209; ¨ p = 0.001); the average number of alleles was 5.04 for Noel S, Ouellet M, Galois P, Lapointe FJ (2007) Impact of urban fragmentation on the genetic structure of the eastern red-backed Danville and 5.21 for Reform. Loci Plal_190b and salamander. Conserv Genet 8:599–606 Plal_EIXNY deviated significantly from expected values of Park SDE (2001) Trypanotolerance in West African cattle and the Hardy–Weinberg equilibrium (Table 2). Loci PG_RIH and population genetic effects of selection. University of Dublin, Plal_545b Dublin were monomorphic in the screened samples, Petranka JW (1998) Salamanders of the United States and Canada. although both are polymorphic across a broader geographic Smithsonian Institution Press, Washington range (WEP, unpublished results). Cross-amplification was Rousset F (2008) GENEPOP’007: a complete re-implementation of successful in 22 of the 27 screened primers with 2–12 the GENEPOP software for Windows and Linux. Mol Ecol alleles (mean = 7.05) and observed heterozygosity from Resour 8:103–106 Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users 0.125 to 1.000 (mean = 0.676). This is only the third and for biologist programmers. In: Krawetz S, Misener S (eds) species of Plethodon, the most species genus of amphibians Bioinformatics methods and protocols: methods in molecular in North America (Collins and Taggart 2009), in which biology. Humana Press, Totowa microsatellite loci have been characterized. Based on our Storfer A, Murphy MA, Spear SF, Holderegger R, Waits LP (2010) Landscape genetics: where are we now? Mol Ecol 19:3496–3514 cross-species amplification with P. shermani, these primers Stuart NS, Chanson JS, Cox NA, Young BE, Rodrigues ASL, appear to have generality within the genus Plethodon, Fischman DL, Waller RW (2004) Status and trends of amphibian making them invaluable tools for gaining a greater declines and extinctions worldwide. Science 206:1783–1786

123